Overview
In addition to the traditional kinase assay that detects phosphorylation of a substrate, you can also use Alpha technology to measure substrate binding to the kinase. This can be useful when screening for inhibitors that block the substrate's interaction with the kinase. The assay is set up so that your kinase interacts with one bead (usually via the use of a tagged kinase with the appropriate affinity bead), while the substrate interacts with the other bead (via the use of a tag or biotin with an appropriate affinity bead). For a list of all catalog bead products, refer to the Alpha products and catalog numbers section below. You can use AlphaLISA™ beads if your interaction needs to be measured in complex matrices, such as a cell lysate, or if you are screening small molecule libraries and would like to minimize possible interference.
Figure 1. An Alpha substrate binding assay, measuring binding of protein substrate to kinase.
What do I need to run this assay?
Required reagents available from Revvity:
- Alpha Donor beads that will interact with one binding partner
- AlphaScreen or AlphaLISA Acceptor beads that will interact with the other binding partner
- Microplates (we recommend our light gray AlphaPlates™, though white plates for fluorescence such as our standard OptiPlates™ and Proxiplates™ also work). Also see Microplate selection.
- TopSeal™-A adhesive plate seal
Required reagents available from various suppliers:
- Kinase (tagged as necessary)
- Substrate (tagged as necessary)
Instrumentation/equipment:
- A plate reader capable of reading Alpha assays
- Optional: plate shaker/rocker
Assay development
Please refer to our Alpha protein-protein and protein-nucleic acid interactions page for assay development considerations.
Tips
- For biomolecular interaction assays, we usually recommend using the weaker affinity beads (Ni-NTA and glutathione beads, rather than anti-GST and anti-His antibody beads) to bind your tagged biomolecules. There is less risk of hooking on the weaker affinity beads.
- The theoretical maximum capacity of a streptavidin-coated bead at 20 µg/mL of bead is 30 nM. Please note that this does not take into consideration the size of the biotinylated molecule that will associate with the bead. For example, you may find that a biotinylated antibody will saturate the bead at 2-3 nM, rather than at 30 nM. If you have saturated the bead, you may see a hook effect.
- The theoretical maximum capacity of an antibody-coated bead is 3-10 nM. Please note this is a theoretical number — you may be able to add more or less than 3-10 nM "antigen" to your assay before hooking, depending on how strong the antibody-antigen interaction is and how large your antigen is.
- The theoretical maximum capacity of a Ni-NTA or glutathione bead for a tagged protein is ~ 30 nM. However, because the His-Ni-NTA and GST-glutathione interactions are weak compared to other interactions (and more tagged protein will be dissociated from the bead at equilibrium), you will likely be able to use 300-1000 nM tagged protein before reaching the hook point.
For research use only. Not for use in diagnostic procedures.
The information provided above is solely for informational and research purposes only. Revvity assumes no liability or responsibility for any injuries, losses, or damages resulting from the use or misuse of the provided information, and Revvity assumes no liability for any outcomes resulting from the use or misuse of any recommendations. The information is provided on an "as is" basis without warranties of any kind. Users are responsible for determining the suitability of any recommendations for the user’s particular research. Any recommendations provided by Revvity should not be considered a substitute for a user’s own professional judgment.